Impact of bread diet on intestinal dysbiosis and irritable bowel syndrome symptoms in quiescent ulcerative colitis: A pilot study

Gut microbiota may be involved in the presence of irritable bowel syndrome (IBS)-like symptomatology in ulcerative colitis (UC) patients in remission. Bread is an important source of dietary fiber, and a potential prebiotic. To assess the effect of a bread baked using traditional elaboration, in comparison with using modern elaboration procedures, in changing the gut microbiota and relieving IBS-like symptoms in patients with quiescent ulcerative colitis. Thirty-one UC patients in remission with IBS-like symptoms were randomly assigned to a dietary intervention with 200 g/d of either treatment or control bread for 8 weeks. Clinical symptomatology was tested using questionnaires and inflammatory parameters. Changes in fecal microbiota composition were assessed by high-throughput sequencing of the 16S rRNA gene. A decrease in IBS-like symptomatology was observed after both the treatment and control bread interventions as reductions in IBS-Symptom Severity Score values (p-value < 0.001) and presence of abdominal pain (p-value < 0.001). The treatment bread suggestively reduced the Firmicutes/Bacteroidetes ratio (p-value = 0.058). In addition, the Firmicutes/Bacteroidetes ratio seemed to be associated with improving IBS-like symptoms as suggested by a slight decrease in patient without abdominal pain (p-value = 0.059). No statistically significant differential abundances were found at any taxonomic level. The intake of a bread baked using traditional elaboration decreased the Firmicutes/Bacteroidetes ratio, which seemed to be associated with improving IBS-like symptoms in quiescent ulcerative colitis patients. These findings suggest that the traditional bread elaboration has a potential prebiotic effect improving gut health (ClinicalTrials.gov ID number of study: NCT05656391).

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Reviewer 1:
1-"Of note: English language check will be needed."

Response:
We appreciate the reviewer's feedback and have carefully reviewed the entire manuscript for language improvements.We believe the changes made have enhanced the overall clarity and readability of the manuscript.

2-"Design and methods:
It is important that the patients with UC in the remission were studied.However, the description of the design is unclear.The authors write the activities in 'both visits', but there is no indication before that if the design is parallel or cross-over design.This should be very clear in the beginning of the explanation of the design.It is suggested that the description is moved as the first chapter in the material and methods -section to allow reader to better follow the flow of the study and decisions made in the recruitment.In addition, it is important that the figure of the design is part of the main document, not as supplement.In dietary interventions the design and justification underlying the choice of the design are elemental to enable clear evaluation of the results.
It is of note that the study may have been improved remarkably if the group of healthy population would have been studied as well.Already in healthy population there is a large variation in the intestinal microbiota composition, and the variability increases along the disease impact, especially in the populations studied here.Thus, it is not possible to really state that the changes would have been caused by the actual bread consumption.
The description of the drop outs should also be included in the methods section.It is a pity that the study ended up with overall number of 23 participants, that does not allow -in practical terms -many conclusion with such a challenging population studied here.
Regarding the bread, it is of interest why the sodium content is clearly higher than in the target bread?There are indications that high salt intake might have an impact on the intestinal microbiota, and thus, might also have an impact here, especially with low number of the participants.
Statistical procedure of the clinical and biochemical measures is quite vague.It is understood that with such a low number of participants one, in principal, should use nonparametric methods or be careful with the normal distribution of the variables.However, the long term study should be analysed fikrst for the time x group -interactions and time point or within group differences.Thus, the analyses such as mixed model time trends should be used to see if there are any differences between the groups.With the Rpackage used here it is fairly simple to do and to present for the reader." Response: We appreciate reviewer 1 comments with respect to design and methods of our study.We have taken the following actions:  We have clarified the study design at the beginning of the sub-section "Study design" (lines 111-114). We have relocated the entire sub-section "Study design" to the beginning of the Methods section.We believe this adjustment will allow readers to better follow the flow of the study. A detailed description of the study design was included as supplementary information (S1 Fig) , providing a comprehensive explanation of our approach.Regarding the figure placement, we would like to clarify that in accordance with PLOS ONE's style requirements, we designated 'Figure 1' for the flow diagram, which includes enrollment schedule, allocation and follow-up.This flow diagram is essential for depicting the study's progression and adherence to protocols.In addition, considering the overall content within the main text, we have already included five figures and three tables.
Given PLOS ONE's formatting guidelines, we would like to ensure that our submission aligns with their requirements while maintaining the clarity and comprehensiveness of our study presentation. We have refined the clarity of our study's aim, which is to examine the potential effects of bread consumption on the symptoms of individuals with coexisting IBS and UC (lines 114-116).We have explicitly highlighted that our study design intentionally focused on this particular patient population, thereby not considering the inclusion of a control group of subjects. We included a description of drop-outs in the "Baseline characteristic" sub-section (lines 318-322) of the Methods, as well as a schematic representation in Figure 1 (flow diagram).We also share the concern about finalizing the study with a total of 23 participants.While we recognize the challenges of working with a limited sample size in this complex population, we have conducted the study to the best of our ability within the constraints of the available participants and acknowledged the limitations of the sample size and its impact on the discussion and conclusions in the manuscript. We have included some words in the Discussion section (lines 473-476) regarding the potential effect of high sodium intake on the intestinal microbiota and the nonsignificant shifts observed in the gut microbiome in our study. We have conducted linear mixed model and generalized mixed model analyses specifically examining the interaction between intervention and time and controlling for individual variability.We have incorporated the results of these analyses into our revised manuscript, specifically in sub-section "Effect of bread diet on clinical symptomatology" of Results and in Table 3 (formerly Table 2 in the previous version of the manuscript).We have also added the statistical approach used in the Methods section (lines 231-239).We believe that these revisions will substantially strengthen the statistical foundation of our study and provide a more comprehensive understanding of the data.

3-"Results:
Baseline characteristics in the whole 31 population and treatment groups based characteristics in the group of 23 participants need to be shown in the main document, not in the supplement.Why are these information that is essential for the reader hidden in the supplement?It is reported that in both treatment groups the same number ( 7) of participants had complete relief of the abdominal pain.What might have caused this impact?It does not support the hypothesis and thus, needs to be carefully discussed.
Give explanations for all the abbreviations as table's footnotes -some of them are missing." Response: To better clarify the findings in the study, we have taken following actions:  We have included baseline characteristics information as Table 2 in the sub-section "Baseline characteristics" in the Results (line 328) to make it more accessible for the reader.In addition, we have added more information in this table regarding other concomitant clinical treatments (i.e., biologic and mesalazine treatments). In our discussion, we considered several potential explanations to the unexpected relief of abdominal pain in both intervention groups (lines 488-495).These factors encompass the similarity in nutritional parameters of both breads despite significant differences in their dough preparation methods, constraints imposed by our relatively small sample size, and the potential influence of uncontrolled environmental variables such as diet, smoking, and weight.In addition, in a previous in vitro study, we observed that the bread baked using modern elaboration proxies (here used as control) also increased the production of short-chain fatty acids by the fecal commensal microbiota present in IBD patients (see reference 38 from the manuscript).We firmly believe that these limitations discussed in the manuscript offer valuable insights into this particular observation. We provided detailed description of unexplained abbreviations as footnotes in Table 3 (former table 2 in the unrevised manuscript)

4-"Discussion:
The authors state at the beginning of the discussion: 'The key finding of this pilot study was that traditional bread intake decreased the Firmicutes/Bacteroidetes ratio, which seemed to be associated with a relief of IBS-like symptoms.However, symptomatology relief was observed in both the treatment and control groups."Was this really the finding?The statistical significance is missing, although near the significance and there was no difference when compared to the control bread group.In addition the relief of symptoms was happening in both groups that is clearly stated.The statement is not convincing based on the present results.In addition, the results related with the diversity have to be summarized also in the beginning of the Discussion.It is appreciated that later in the Discussion it is clearly written that other factors explain the differences found in the diversity measures." Response: We appreciate Reviewer 1 insights.We have clarified the key finding statement in the Discussion section to better align with the results (lines 436-437).We hope these changes enhance the clarity and accuracy of our manuscript.

5-"Limitations:
This kind of dietary study cannot really be double blinded -breads will differ either in their appearance and or their taste.So this statement needs to be discarded.As the study is introduced as the pilot study, how the results seem for the authors?Do these results indicate the start of the main study?This could be clearly discussed and justified in addition to the usual text in the conclusion regarding the need of further studies." Response: We took specific measures to minimize these differences, ensuring that both types of bread had similar appearance and taste when supplied to participants.By doing so, our intention was to prevent both participants and investigators/caregivers from distinguishing between the two types of bread based on taste or appearance, thus avoiding a predisposition to change by study subjects.We strongly believe that the double-blinding is one of the strengths of the present study.
We revised our conclusions and made some adjustments to highlight the preliminary nature of this study and the need for subsequent investigations (lines 515-522).
Reviewer 2: 1-"Baseline Difference in Diets: Please provide more detailed information regarding the baseline dietary habits of your study participants, as differences in diets can influence gut microbiota and may confound your results." Response: Thank you for your comments and for revising the manuscript.While we agree that differences in diets can influence gut microbiota and may potentially confound our results, we focused on assessing the impact of the specific bread-based dietary intervention on the modification of UC with IBS-like symptomatology.As such, we did not collect extensive data on participants' dietary habits beyond the study's dietary intervention and adherence to Mediterranean diet.However, participants were asked to not alter their diet during the intervention period (lines 120-121).

2-"Clinical vs. Endoscopic vs. Deep Remission:
Consider discussing the differences and implications of clinical, endoscopic, and deep remission, as these distinct states may have varying effects on gut microbiota and symptomatology.Were all recruited patients only in clinical remission, or did some have endoscopic and histological remission as well, and how does this impact the findings?" Response: In this study, our primary objective was to investigate the effects of a traditional bread-based dietary intervention on the gut microbiota and symptomatology in UC patients in clinical and endoscopic remission, defined as a total Mayo score of lower than 2. We have clarified this along the new manuscript.By focusing on this specific remission state, we aimed to create a targeted and homogeneous cohort that would facilitate recruitment and study design.However, we acknowledge the potential variations in microbiota and symptomatology associated with other remission states, such as histological or deep remission.This study lays the foundation for future research that can delve into the distinctions between these remission states to gain a more comprehensive understanding of their effects.

3-"Impact of Lockdown and COVID-19:
Since the study was conducted during lockdown and the COVID-19 pandemic, please address whether any participants had COVID-19 during the trial, as COVID-19 and its treatment could potentially impact gut microbiota and symptoms." Response: We confirm that none the participants reported experiencing COVID-19 symptomatology or testing positive for COVID-19 during the trial.This aspect was not explicitly stated in the initial version of the manuscript, and we have now added a statement in the methodology section to clarify this (line 329).

4-"Lack of Detailed Bread Composition:
Provide a comprehensive analysis of the bread's composition, including fiber content and prebiotic components, to elucidate the dietary factors influencing the gut microbiota." Response: We did collect data on the nutritional composition, including fiber content, for both types of bread, and this information is provided in Table 1.However, we acknowledge that a more detailed analysis of prebiotic components would have been beneficial, and we plan to consider this in future research.We appreciate the feedback and will ensure that future investigations provide a more in-depth assessment of dietary factors.Moreover, we have identified an error in the description of the components involved in modern bread production.
We have subsequently refined this definition to specify that modern bread incorporates a minimal proportion of sourdough starter (line 203).We would like to emphasize that this change is purely a clarification of terminology and does not impact the core content, findings, or discussion presented in the manuscript.

5-"Short Duration:
Eight weeks may be too short of a follow-up period and might not reflect the sustainability of the microbiota changes, as short follow-up may be one of the limitations of this study." Response: We have expanded our discussion (lines 498-499) to note that longer-duration studies could be favorable for the identification of more substantial microbiome changes.We selected an 8-week duration based on findings from previous studies (e.g., Dong TS et al., 2020;Nutrients), expecting it to be sufficient to observe microbiota changes in response to the dietary intervention.However, longer diet durations may be necessary to identify major microbiome shifts.
6-"Discussion of Non-significant Findings: Provide a more in-depth discussion of non-significant findings, explaining their potential biological relevance and considering the study's statistical power."